Expression of plant genes is controlled in a complex pattern during the life cycle of a plant. Several processes are involved in this regulation of gene expression. The main steps are; the initiation of transcription; the termination of transcription; the processing of transcripts; the transport of mRNA to the ribosomes; and the translation.
Three examples of plant genes that are expressed are found in Huang et al (1996 Biosci Biotech Biochem 60 (2) 233–239) who report on three rice sucrose synthase isogenes, which are called RSus1, RSus2 and RSus3. The authors also report on their differential regulation of their expression. The authors state that the gene organisation patterns of RSus2 and RSus3 were the same.
One of the major processes controlling gene expression is initiation of transcription. The transcription is initiated by binding of RNA polymerase together with several transcription factors to the promoter region. Specific regulatory DNA sequences (cis-elements) in the promoter serve as binding sites for these transcription factors (trans-acting factors).
The cis-elements found in plant gene promoters can be divided in two categories. The first category comprises those cis-elements which are involved in initiation of transcription. The TATA box and the CAAT box are examples of proximal cis-elements involved in initiation of transcription. The CAAT box defines the binding site for the RNA polymerase, and the TATA box directs the RNA polymerase to the correct transcription start site. Presence of multiple CAAT boxes normally indicate a constitutive promoter. The TATA box and CAAT box are conserved among prokaryotes and eukaryotes, but are not essential for the function of some plant gene promoters. The second category is composed of cis-elements which are involved in temporal and spatial regulation of gene expression. Genes encoding seed storage proteins (Glutamins, Legumins, Prolamins etc.) are examples of genes which are temporally and spatially regulated, and are thereby expressed in a tissue-specific and developmental manner. Examples of endosperm-specific cis-elements are the AACA motif and the endosperm box. These cis-elements, that contribute to tissue-specific and developmental expression of endosperm storage protein genes, are conserved among a wide range of seed storage protein genes.
The manner in which complex patterns of transcription factors act on specific cis-elements determines whether genes are more or less constitutively expressed, or are expressed at specific times during development. Furthermore, these interactions determine whether expression occurs in a specific tissue e.g. the endosperm, is displayed by several tissues e.g. those of the seed, or is common to all parts of the plant. Multiple trans-acting factors can recognise variants of the cis-element consensus sequences and compete for binding, yielding complex expression patterns. Each plant gene promoter has a set of transcription factors and other trans-acting factors, which interact with the promoter sequence and the RNA polymerase and thereby regulate gene expression in an unique pattern.
It is known that it is desirable to direct expression of a nucleotide sequence of interest (“NOI”) in certain tissues of an organism—such as a plant. The NOI will typically encode a product of interest (“POI”). For example, it may be desirable to produce crop protein products with an optimised amino acid composition and so increase the nutritive value of the crop. It may even be desirable to use the crop to express non-plant genes such as genes for mammalian products. Examples of the latter products include interferons, insulin, blood factors and plasminogen activators.
However, whilst it may be desirable to achieve expression of a NOI in certain tissues it is sometimes important (if not necessary) to ensure that the NOI is not expressed in other tissues in such a manner that detrimental effects may occur. Moreover, it is important not to upset the normal metabolism of the organism to such an extent that detrimental effects occur. For example, a disturbance in the normal metabolism in a plant's leaf or shoot could lead to stunted growth of the plant.
An example of the use of plant promoters to cause expression of an NOI in plant tissue may be found in CA-A-2006454, which describes a DNA sequence of an expression cassette in which the potato tuber specific regulatory regions are localised. The expression cassette contains a patatin-gene with a patatin-gene promoter. The DNA sequence is transferred into a plant genome using Agrobacterium. According to CA-A-2006454, the DNA sequence enables heterologous products to be prepared in crops.
However, in plant transformation processes, it is generally the low efficiency of both transformation and regeneration that seriously slow vector development, since they limit the number of genetic constructs which can be tested. Investigations of the strength and tissue specificity of different transcriptional promoters, which can greatly influence the effect of the genetic manipulation, can be unmanageably labour-intensive if performed in stable transformants. This has resulted in a tendency to use strong constitutive promoters which are not tissue-specific to direct transgenic expression in stable transformants, e.g. from viruses (cauliflower mosaic virus 35S promoter) and Agrobacterium (nopaline synthase promoter (NOS)). Not the least problematic element of this approach occurs when lowering of gene expression by antisense transcription is attempted, because gratuitous suppression of gene expression in all tissues can result in developmental retardation of non-target tissues, or other weakening of the transformant. Under such circumstances, only those transformants, in which the genetic construct poorly suppresses gene expression, would be expected to survive selection. There is, therefore, a strong argument for the use of tissue-specific transcriptional promoters for directing antisense transcription, but, in many crop species, characterising such promoters in stable transformants is not feasible owing to low efficiency of transformation and regeneration.
Despite the fact that there are already some promoters available in the art, there is still a need to have additional promoters, in particular promoters that are efficient and/or selective in their ability to allow for the expression of a NOI.
Thus, the present invention seeks to provide a promoter that is capable of causing the expression (transcription) of a NOI.
More in particular, the present invention seeks to provide a promoter that is capable of directing the expression (transcription) of a nucleotide sequence of interest in specific tissues, or in just a specific tissue, of an organism, typically a plant.